Cancer is characterized by genetic instability both at the chromosomal level and at the nucleotide level. The acquisition of certain mutations confers a selective advantage to the cancer cells and is critical in cancer progression. A diverse array of defects in both DNA polymerases and DNA repair enzymes appears to contribute to the increased genetic instability observed in cancer cells (Hanahan et al, 2011, Cell, 144: 646-674). Although necessary to confer a selective advantage to cancer cells, excessive instability in the cancer cell genome is suggested to be incompatible with cell viability. Treatment of cancer by increasing the genetic instability of cancer cells beyond the threshold over which the cancer cells are no longer viable has been proposed as an alternative therapeutic approach (Loeb, 2011, Nature Reviews Cancer 11, 450-457).
A variety of anticancer drugs, including platinum drugs, alkylating agents, and anthracyclines, share DNA as a common target of biological activity. Covalent binding of drugs to DNA or other interactions that interfere with transcription and/or replication initiates a series of events that, although intended to rescue the cell for further proliferation, may eventually lead to cell death. This depends on various factors, including the degree of drug binding, the activity of the DNA repair systems, and the balance between pro- and anti-apoptotic mechanisms in the cell. The cytotoxic effect, and therefore the clinical effectiveness, of these classes of drugs can potentially be reduced by the action of DNA repair enzymes and damage-signal molecules. In contrast, if DNA repair is deficient, a phenotype which may contribute to malignant progression (mutator phenotype), the resulting tumour may be more susceptible to DNA-damaging agents (1). One deficiency that contributes to oncogenesis but leaves the tumour vulnerable to targeted treatment directed against other genes or gene products capable of compensating for the original deficiency is referred to as “synthetic lethality.”
Synthetic lethality (also known as Synthetic Sickness/Lethality or “SSL”) can be described as follows: “Two genes have a SSL relationship when inhibition or mutation of either gene alone does not cause loss of viability/sickness, but simultaneous inhibition of both genes results in reduced cell viability or an impairment of cellular health/fitness.” (Brough et al, 2011, Curr Op in Gen and Dev., 21: 34-41). Brough et al. also describe how SSL relationship can be used to identify therapeutic options in that if one gene in an SSL relationship is a tumour suppressor gene, then its synthetic lethal partner becomes a candidate therapeutic target for tumours with a defined tumour suppressor gene dysfunction. SSL can occur between genes acting in the same biochemical pathway or in distinct but compensatory pathways, and components of the same pathway often share the same SSL partners. Synthetic lethality can be mimicked by targeted therapies (Chan et al, Nat. Rev. Drug. Discov., 10: 351-364).
Two DNA repair-associated proteins that are known to be deficient in several forms of inherited cancer susceptibility are BRCA1 and BRCA2. These proteins are intimately involved with proteins such as PALB2 and RAD51 in mediating homologous recombination (HR)-dependent DNA (HR-DD) repair, the most precise of several repair pathways (8-10). BRCA2 mediates binding of RAD51 to short, single-stranded DNA as part of the recognition of DNA strand breaks and initiation of DNA repair (8, 9). BRCA1 is then involved in processing of the free end of a DNA strand break, whereas BRCA2 is essential to a strand-exchange step of HR (9). The repair complex includes direct physical interactions between BRCA1, BRCA2, PALB2, BARD1 and RAD51, not only at sites of DNA damage but also at chromosomal foci in mitotic cells (8).
Another important protein in mediating base-excision DNA repair (single strand DNA break repair) is the enzyme PARP1 (poly[adenosine diphosphate (ADP)-ribose] polymerase) (8). PARP regulates transcription of genes involved in other repair mechanisms, including BRCA2 (11). However, PARP is also involved in repair pathways that are independent of BRCA1 and BRCA2 pathways and that tend to be more error-prone (8). If cells are deficient in BRCA1 or BRCA2, and thus HR-DD repair, the cells become dependent upon PARP-dependent repair pathways (8). In this case, repair is very sensitive to inhibitors of PARP, and cells tend to accumulate replication-generated errors that would normally be repaired immediately, leading eventually to cell cycle arrest and cell death (8).
Clinical examples of drug-hypersensitivity of DNA-repair-deficient tumours have been described. Familial carcinomas of breast, ovary and prostate with a deficiency of BRCA1 or BRCA2 are more sensitive to olaparib (4-[(3-{[4-cyclopropylcarbonyl)piperazin-1-yl]carbonyl}-4-fluorophenyl)methyl]phthalazin-1(2H)-one; also known as AZD2281). Treatment of BRCA1- or BRCA2-deficient tumours with olaparib has resulted in good clinical responses (2).
As a method to study the function of specific gene products in cellular processes, researchers have utilized the ability of nucleic acid that is complementary to mRNA to initiate degradation of that mRNA specifically. This phenomenon, which exists in cells as part of a stringent mechanism of control of mRNA levels as well as an antiviral defence, makes use of nucleic acids that are referred to as “antisense”. Specific down-regulation of intracellular proteins can be accomplished with the use of such antisense nucleic acids that bind specifically, based on sequence matching and Watson-Crick base-pairing, to a selected mRNA target. By recruitment of intracellular endonucleases, the target mRNA is destroyed, and the protein usually generated from it disappears with normal turnover. Full-length antisense mRNA expression vectors are currently not of potential clinical use. However, shorter antisense nucleic acids have clinical potential, and one format has already been used in clinical trials. Several different chemistries of antisense molecules have been used in experimental systems to specifically down-regulate an mRNA of interest. Oligonucleotides (OLIGOs) consist of a single-stranded molecule that is introduced into cultured cells using a cationic liposomal transfecting agent in order to permeate the cell membrane, although there is some indication that carriers in the blood are able to facilitate entry of OLIGOs into cells in vivo.
It has been reported that down-regulation of BRCA2 using an antisense OLIGO targeted against the region of the translational start site increased the sensitivity of tumour cells to mitoxantrone in vitro (6). The authors concluded that these effects could be applied to a BRCA2 genetic screening method as a predictor of response to a specific therapeutic approach. It has also been reported that cells treated with a pool of 4 siRNAs targeting BRCA1 or BRCA2 (Dharmacon, Thermo Fisher, Lafayette, Colo., USA) were more sensitive than control siRNA-treated counterparts to cytotoxic activity of a PARP inhibitor (7). In this study, the authors concluded that their synthetic lethal siRNA screen with chemical inhibitors could be used to define new determinants of sensitivity and potential therapeutic targets. Both of these two studies focussed on the use of BRCA2 inhibition in screening methods; neither suggested that inhibition of BRCA2 may have therapeutic potential.
U.S. Pat. No. 5,837,492 describes materials and methods used to isolate and detect a human breast cancer predisposing gene (BRCA2) and describes generally polynucleotides comprising all or a part of a BRCA2 locus, including antisense oligonucleotides.
United States Patent Publication No. 2004/0097442 describes compounds, compositions and methods for modulating the expression of BRCA2 region transcription unit CG005, which is a region of the BRCA2 locus that is outside the BRCA2 gene itself. The compositions comprise oligonucleotides targeted to nucleic acid encoding BRCA2 region transcription unit CG005, i.e. oligonucleotides targeted to mRNA encoding part of the BRCA2 locus other than the BRCA2 gene. Methods of using these compounds for the diagnosis and treatment of disease associated with expression of BRCA2 region transcription unit CG005 are also generally described. United States Patent Publication No. 2004/0097442 does not describe antisense oligonucleotides directed to the mRNA encoding BRCA2.
United States Patent Publication No. 2005/0227919 describes methods and means relating to the treatment of cancers which are deficient in HR-dependent DNA DSB repair using inhibitors which target base excision repair components such as poly (ADP-ribose) polymerase (PARP).
International Patent Application No. PCT/EP2007/008852 (Publication No. WO 2008/043561) describes pharmaceutical compositions comprising modulators of kinases, kinase-binding polypeptides and/or an inhibitor for influenza virus replication for the prevention and/or treatment of influenza. This application also describes genome-wide screening to identify human genes that are relevant for replication of influenza viruses. Several thousand genes were identified, including BRCA2, and target sequences for “knocking down” each gene using siRNA technology were also identified. Four target sequences within BRCA2 were identified.
United States Patent Publication No. 2011/0230433 describes methods and composition for treatment of cancer by increasing the mutation rate of cancer cells beyond an error threshold over which the cancer cells are no longer viable.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.